Human skin is relatively simple tissue that performs varied and complex functions such as temperature regulation, the processing of vitamin D precursors, the excretion of urea, and the storage of carbohydrate and fat. The skin contains many unique cell types to effect its specialized functions. On a gross level, the skin consists of epidermis and a basement membrane zone overlying dermis and subcutaneous fat. Skin tissue arises embryologically from ectoderm, neuroectoderm, and mesoderm. The epidermis, hair and sebaceous glands (pilosebaceous units), sweat glands (eccrine units), and nails are all ectodermal derivatives. Neuroectodermal derivatives include melanocytes, nerves, and special neuroreceptors, while mesenchymal derivatives include collagen, reticulin and elastic fibers, blood vessels, muscle, and fat. Probably the most important function of the skin is that of protection; the skin produces a scaling surface impermeable to many substances, has an elaborate network of immunocompetent cells constantly monitoring for potentially harmful antigens, and produces pigment which filters out harmful rays of ultraviolet radiation from the sun.
Ultraviolet radiation is a major environmental damaging agent causing photodamage to the skin, including cutaneous malignancies and photoaging (see generally, Fitzpatrick's Dermatology in Medicine, Fifth Edition, I. M. Freedberg, et al., eds., McGraw-Hill (1999)). Clinical features of photoaging include wrinkles, skin laxity and coarseness, and pigmentation disorders. While histological manifestations of photoaging have been well known for some time, the molecular mechanisms that cause them have only recently become a focus of concerted studies.
Solar light contains a broad spectrum of energy wavelengths, and ultraviolet radiation which is among the relatively short wavelengths occurring at between 100 and 400 nanometers (in contrast to the visible light spectrum which occurs at between about 490 and 690 nanometers). Ultraviolet radiation is composed of three segments, designated as A, B, and C. Ultraviolet-C radiation (between 100 and 280 nanometers) is filtered out by the earth's ozone layer and is not known to pose a health threat. There is evidence, however, that exposure to both ultraviolet-A radiation (between 315 and 400 nanometers) and ultraviolet-B radiation (between 280 and 315 nanometers) can have adverse short-term and long-term effects on skin health and visual health. For example, ultraviolet radiation is known to play an important a role in both the development of skin cancers and premature aging of the skin.
The cell type most affected by ultraviolet radiation in humans is the keratinocyte. When illuminated by ultraviolet radiation, the keratinocyte reacts in three generally non-overlapping ways. First, it initiates a DNA repair response, which is activated by the DNA damage itself (Herrlick et al. (1994) Adv. Enzyme Reg. 34:381–95). Second, it signals to the surrounding tissue by releasing pro-inflammatory cytokines, such as IL-1 and TNFα (Ullrich et al. (2000) J. Dermatol. Sci. 23:S10–2; Ouhtit et al. (2000) Am. J. Pathol. 156:201–7; and Beissert et al. (1999) J. Investig. Dermatol. Symp. Proc. 4:61–4). Third, the keratinocyte activates its inherent responses to ultraviolet radiation by changing its physiology, including regulation of gene expression, cytoskeletal rearrangements, and induction of apoptosis (Zhuang et al. (2000) J. Interferon Cytokine Res. 20:445–54; and Assefa et al. (1997) J. Invest. Detmatol. 108:886–891).
The inherent responses of keratinocytes to ultraviolet radiation, by analogy with responses to other extracellular signals, can be separated into two phases, the immediate and delayed. The immediate phase contains the ultraviolet radiation-specific signal transduction cascades and results in activation of transcription factors. In the delayed phase one sees the changes in gene expression. The invention herein provides a characterization of the ultraviolet radiation-responsive induced and suppressed genes in human epidermal keratinocytes.
The histological signs of photoaging at the epidermal level include the following: (1) variation in the thickness of the epidermis (atrophy or hyperplasia according to the zones observed); (2) a cellular atypia (Kligman et al. (1986) Photodernatol. 3:215–227); (3) a loss of cell polarity; (4) an unevenness of the horny layer; (5) a reduction in the number of Langerhans' cells (Lavker et al. (1987) J. Invest. Dennatol. 88:44s-51s); (6) a pigmentation characterized by a mosaic appearance with hypo- or hyperpigmentation zones; and (7) a linearization of the dermo-epidermal junction (Lavker (1979) J. Invest. Dermatol. 73:59). For a review of photoaging of the skin, see Gilchourest, Skin and Aging Processes, 1989, CRC Press.
The biologic responses of cells exposed to ultraviolet radiation have been studied in a wide variety of systems, from Esherichia coli to man. In humans, the molecular effects of ultraviolet radiation include DNA damage, apoptosis and activation of the Jun N-terminal kinase (JNK) and the nuclear factor kappa-beta (NFkB). Both studied recently, although not extensively in epidermal keratinocytes, which are the primary target of ultraviolet radiation. A major impetus for studies of the molecular response to ultraviolet radiation came with the identification of the protein kinase that bound to and activated the c-Jun transcription factor in response to ultraviolet radiation (Derijard et al. (1994) Cell 76:1025–1037). The kinase was named “JNK” for Jun N-terminal kinase, or “SAPK” for stress activated protein kinase (Kyriakis et al. (1994) Nature 369:156–160). Soon it was realized that JNK responds to several extracellular signals in addition to ultraviolet radiation, such as osmotic shock, arsenate, and pro-inflammatory cytokines (Rosette et al. (1996) Science 274:1194–7; Cavigelli et al. (1996) E.M.B.O. Journal 15:6269–79). JNK can phosphorylate additional transcription factors, including Elk1 and ATF2 (Kallunki et al. (1996) Cell 87:929–39). JNK is itself activated by a small number of relatively specific kinases, designated “JNKKs.” Many kinases, designated JNKKKs, respond to a large variety of stimuli to phosphorylate and activate “JNKKs;” the ultraviolet radiation-responsive JNKKK has not yet been identified (Fanger et al. (1997) Curr. Opin. Genet. Dev. 7:67–74).
Another clear molecular effect of ultraviolet radiation is the activation of the NFkB transcription factor (Devary et al. (1993) Science 261:1442–5). The activation of NFkB by ultraviolet radiation is not associated with DNA damage and occurs even in cytoplasts devoid of nuclear DNA (Devary et al. (1993) Science 261:1442–5; Simon et al. (1994) J. Invest. Dermatol. 102:422–7). Inactive NFkB resides in the cytoplasm complexed with IkB protein. Upon activation by a very large and varied set of extracellular stimuli, IkB is phosphorylated and thus designated for proteolysis. This results in the release of NFKB, which is then free to enter the nucleus and activate gene transcription (Barnes et al. (1997) NE J. Med. 336:1066–71). The ultraviolet radiation-responsive kinases that mark IkB for degradation have not yet been identified (Li et al. (1998) Proc. Natl. Acad. Sci (USA) 95:13012–13017).
Methods to evaluate photodamage to skin or to cells contained therein have been described in the art. For example, U.S. Pat. No. 6,079,415 provides methods and markers useful for establishing ultraviolet-A radiation damage to the dermis, and more specifically, to the production of collagen by fibroblasts of the dermis. Moreover, methods useful for the prevention of ultraviolet radiation damage to the skin or cells contained therein have been described. For example, U.S. Pat. No. 5,908,836 provides methods for protecting skin from ultraviolet radiation damage using sulphated sugars, and U.S. Pat. No. 5,916,880 provides a method to reduce wrinkles by treatment with sulfated sugars. Numerous other examples have been described in the field. For example, U.S. Pat. No. 5,939,457 describes the use of a hydroxy acid product useful for the reduction of wrinkles, U.S. Pat. No. 5,939,082 describes the use of a vitamin B compound for the regulation of signs of skin aging, and U.S. Pat. No. 5,962,534 describes the use of certain retenoids.
However, studies to date examining the response at the molecular level of skin and the specialized skin cells to exposure to ultraviolet radiation almost exclusively have been limited to the examination of one or several genes and/or proteins specific to a particular cellular process. For example, the study by Garmyn et al. ((1991) Lab. Invest. 65:471–478) describes an immediate and early temporal pattern of keratinocyte response to exposure to ultraviolet radiation, but this study was limited to an analysis of the expression patterns of only a few genes. Moreover, no study has successfully provided an analysis of the complete response of the skin and/or the specialized cells of the skin to exposure to ultraviolet radiation. For example, while the study by Abts et al. ((1997) Photochem. Photobio. 66(3):363–367) utilized the methodology of mRNA differential display to study alterations in gene expression mediated by ultraviolet radiation exposure, the study identified only a very small number of ultraviolet radiation-regulated genes due to the difficulty of the technique used.
The role of the ultraviolet-B radiation has been clearly demonstrated in the induction of ultraviolet radiation-induced skin cancers. It has, as a principal chromophore, nucleic acids and, in particular, deoxyribonucleic acid, in which it induces lesions and/or mutations (Eller, in Photodamage, pp. 26–56, Blackwell, ed. (1995)). In addition, ultraviolet-B radiation has been linked to premature aging of the skin, characterized by a dry, rough clinical appearance associated with a loss of elasticity, as well as marked wrinkles.
Gene array technology is a powerful, new technique for gene expression monitoring, enabling a global view into changes of expression for an extremely large set of genes. The technology has been applied to the study of many biologic processes (see, e, Lockhart et al. (1966) Nat. Biotechnol. 14:1675–1680; Johnston (1998) Curr: Biol. 8:171–174). For example, the technique has been used in the study of cancer (Scherf et al. (2000) Nat. Genet. 24:236–44; Ross et al. (2000) Nat. Genet. 24:227–35; Welford et al. (1998) Nuc. Acids Res. 26:3059–3065; Alon et al. (1999) Proc. Natl. Acad. Sci. (USA) 96:6745–6750; and Golub et al. (1999) Science 286:531–537); for the study of complex pathways of gene expression (Fambrough et al. (1999) Cell 97:727–741; and Galitski et al. (1999) Science 285:251–254); for the study of the aging process (Cheol-Koo Lee (1999) Science 285:1390–1393; Ly et al. (2000) Science 287:2486–92; Harkin (1999) Cell 97:575–586); and for the study of the stress response of cells to particular damaging agents (Jelinsky et al. (1999) Proc. Natl. Acad. Sci. (USA) 96:1486–1491)).
Pharmacological agents useful in the treatment of photodamaged skin have been identified. For example, the normal repair processes in photodamaged skin have been enhanced pharmacologically. The first to be assessed for this property was Tretinoin (all-trans-retinoic acid). Studies have demonstrated that the reconstruction zone of new collagen was significantly deeper in tretinoin-treated mice, with the enhanced repair being dose and time related. In addition, new collagen was histochemically, ultrastructurally, and biochemically normal. As determined by radioimmunoassay, collagen content was increased two-fold, and mRNAs for types I and III collagen were increased two- to threefold in the tretinoin-treated skin. New collagen synthesis was localized with immunofluorescence techniques in the histologically defined reconstruction zone, and the presence of new elastin and increased fibronectin were also established in the region. Isotretinoin (13-cis-retinoic acid) has also been demonstrated to enhance dermal repair in mice. This repair activity remains retinoid-specific. (Fitzpatrick's Dermatology in Medicine, Fifth Edition, I. M. Freedberg, et al., eds., McGraw-Hill (1999), pp. 1717–1721).
Studies at the molecular level have shown that ultraviolet-B radiation up-regulates the collagen-degrading enzymes collagenase and gelatinase, and that tretinoin reduces the mnRNA's, protein, and activities of these enzymes by 50 to 80 percent. Id. at 1721. Thus, pharmacological agents may be used to reverse skin damage resulting from altered expression of proteins and nucleic acid molecules due to ultraviolet radiation exposure.
Because exposure of specialized cells of the skin to ultraviolet radiation plays an important role in the generation of skin cancer and in the process of premature aging, it would be beneficial to characterize at the molecular level the full response of the skin to exposure to ultraviolet radiation. The present invention provides such information, which was previously lacking in the field. Moreover, epidermal keratinocytes, the main target of environmental ultraviolet radiation, have seldom been used as the model system. The invention described herein redresses this deficiency in the art. Furthermore, nucleic acid molecules and protein molecules in skin cells that are regulated by ultraviolet radiation and the methods provided herein are useful resources for the identification of pharmacological agents for the prevention and treatment of skin cancer and premature aging of the skin.